Since radioactive microspheres have been used to map the time-course of development of coronary collateral flow, we tested the stability of microspheres in ischemic tissue. Microspheres were injected prior to coronary artery occlusion in dogs and microsphere estimates of flow to the central ischemic zone were compared with microsphere estimates of flow to the normal zone. We found that preocclusion flow to the central ischemic zone was consistently underestimated by the microsphere technique in dogs sacrificed at 24 hr, 48 hr, or 8 days after permanent coronary occlusion. Thus, microspheres were lost from ischemic tissue. We conclude that microsphere estimates of collateral flow may be inaccurate when occlusion is maintained for 24 hr or more.